Protein-protein interactions play an important role in regulating the physiological functions in cells, such as gene expression, transport, signal transduction and cell cycle control. Identification of the interacting protein partners and the contact sites involved can facilitate the understanding of protein functionalities and assist in identifying and providing novel approaches for the development of treatment and diagnostic methods and agents. Due to the fact that many interactions between proteins are transient, however, the general techniques for elucidating the three-dimensional structure of the complexes, such as X-ray and NMR, are not particularly useful due to technical difficulties. Alternative biochemical approaches need to be developed for structure—function studies of protein-protein interactions.
Crosslinking reagents arc promising tools for studying protein-protein interactions (e.g., Freedman, R. B. Mends Biochem. Sci. 1979, 193-197; Herrmann, et al. Methods Cell Biol. 2001, 65, 217-230; Fancy, D. A. Curr. Opin. Chem. Biol. 2000, 4, 28-32; Fasold, et al. Angew. Chem. Internat. Edit. 1971, 10, 795-801). Crosslinking reagents are, in general, small organic molecules containing two chemical groups (bifunctional) that react with the functional groups on the side chains of proteins. Proteins in proximity to one another can be connected covalently via crosslinking reagents. There are two types of crosslinking reagents. The first type is photoactivable crosslinkers, which can be incorporated by photolysis. The second type is chemical crosslinkers, which can be incorporated under particular chemical conditions. Covalent crosslinking using photoactivable reagents is a preferred method for studying transient protein-protein interactions due to their highly reactive and non-specific insertion properties with any proximal C—H bond.
Khorana's laboratory has been at the forefront of developing useful photocrosslinking strategies for studying the structure and function of protein-protein interactions at the molecular level, using rhodopsin as a model system over the last few decades. For example, the crosslinking reagent 3-(4-(((4-nitro-3-carboxyphenyl)dithio)methyl-t)-phenyl)-3-(trifluoromethyl)-3H-diazirine (DTDA, FIG. 7a; Resek, et al. J. Org. Chem. 1993, 58, 7598-7601) was designed with several specific features for studying rhodopsin-transducin interactions. DTDA is able to form a disulfide bond with accessible cysteines. In combination with site-specific rhodopsin mutants, DTDA can be targeted to unique positions in the protein. In addition, following the formation of carbene, the radioactive label can be transferred to the site of insertion after cleavage of the disulfide bond. This chemical has been applied successfully to the determination of the binding subunit of transducin (Resek, el. al. Pro. Natl. Acad. Sci. USA. 1994, 91, 7643-7647) using gel electrophoresis and fluorographic visualization. However, DTDA is not widely used due to the difficult synthetic procedures and the radioactivity.
In order to characterize the contact sites of the interacting proteins, this method has been developed further in Khorana's lab to include protcomic digestion, streptavidin/biotin purification of peptide fragments and mass spectrometry. A commercially available nitrene-generating arylazide, N-((2-pyridyldithio)ethyl)-4-azido salicylamide (PEAS, FIG. 7b) was used (Cai, et. al. Proc. Natl. Acad. Sci. USA. 2001. 98, 4877-4882). This method has several drawbacks. i) The data is uncertain and crosslinking is inefficient due to the use of an inferior photocrosslinking reagent. Photolysis of simple arylazide releases singlet nitrene, which can isomerize rapidly (10-100 ps) to strongly electrophilic species (benzazirine and cycloheptatetraenes) and undergo bimolecular reactions at room temperature (Gritsan et al. J. Am. Chem. Soc. 2001, 123, 1951-1962). This reaction results in the crosslinking of amino acids that are not in the vicinity of contact sites. Simple arylnitrene also cannot insert into non-activated C—H bonds and results in a very low yield of crosslinking products. ii) Current methods do not provide a means for the efficient capture and elution of crosslinked products for mass spectrometric analysis. Photocrosslinking generally produces heterogeneous crosslinked products at low concentrations: Presently, the most commonly used purification handle for crosslinked products is the sulfhydryl functional group, through which biotin is introduced. This biotin molecule allows the non-covalent capture of crosslinked products through immobilized avidin, and the crosslinked products are eluted out with a large excess of biotin. In such non-covalent capture systems, however, the trapped crosslinked products may be diluted or lost during the washing steps. The presence of a large excess of biotin suppresses the mass sensitivity. iii) A third drawback is due to issues associated with sulfhydryl compounds. Thiol chemistry is not orthogonal to peptide chemistry. Any Cys residues on a protein have to be blocked before the introduction of a biotin moiety. Blocking adds an extra step and complicates data analysis. Also, when working with a very dilute solution (e.g., in the femtomole range), tryptic fragments originating from any contaminates, such as cytokeratin (from the hair), that carry a Cys may react with biotin and interfere with the mass spectrum data. Finally, to prevent the oxidation of free thiol, a reducing/inert environment must be maintained throughout the process. Ideally the purification handle should be orthogonal to peptide functional groups.
This general methods have been further improved in Khorana's lab by Y. Huang using a newly synthesized DTDA analog compound, N-(2-(4-nitro-3-carboxyphenyl)dithioethyl)-4-(3-(trifluoromethyl)-3H-diazirine-3-yl)-benzamide (NETDB, FIG. 7c) and a fluorescence tag purification strategy (FIG. 8). HPLC is used to purify crosslinked peptides digested by trypsin (Huang, Y., Khorana, H. G. “Mapping of Contact Sites in Interaction between Transducin and Light-Activated Rhodopsin.” 17th Symposium of the Protein Society, Jul, 26-30, 2003, Boston, Mass.). The photocrosslinking yield is increased substantially and the crosslinked subunit can be easily detected by fluorescence imager (FIG. 9). Some tryptic peptides have been identified. However, no contact sites have yet been identified on the molecular level.
A few issues remain to be solved if a general approach is established for structural determination of interacting proteins at the molecular level: i) new and efficient crosslinking reagents for capturing the interacting proteins; ii) crosslinking reagents with a variable spacer for determining the distance between the contact sites; iii) method for separation, purification, and sample enrichment to enhance detection, for example, by mass spectrometric analysis; iv) multiple detection methodologies; v) a method for detecting protein-protein interactions in a system that does not require proteins to be purified; and vi) the replacement of cleavable disulfide linkages.